Structure-Activity Studies of Nitroreductase-Responsive Near-Infrared Heptamethine Cyanine Fluorescent Probes

Abstract

Two new classes of near-infrared molecular probes were prepared and shown to exhibit "turn on" fluorescence when cleaved by the nitroreductase enzyme, a well-known biomarker of cell hypoxia. The fluorescent probes are heptamethine cyanine dyes with a central 4'-carboxylic ester group on the heptamethine chain that is converted by a self-immolative fragmentation mechanism to a 4'-caboxylate group that greatly enhances the fluorescence brightness. Each compound was prepared by ring opening of a Zincke salt. The chemical structures have either terminal benzoindolinenes or propargyloxy auxochromes, which provide favorable red-shifted absorption/emission wavelengths and a hyperchromic effect that enhances the photon output when excited by 808 nm light. A fluorescent probe with terminal propargyloxy-indolenines exhibited less self-aggregation and was rapidly activated by nitroreductase with large "turn on" fluorescence; thus, it is the preferred choice for translation towards in vivo applications.

Keywords: NIR fluorescence; cell microscopy; cyanine dye; enzyme; molecular sensor.

Scheme 1

(a) Reduction of the probe’s nitro group by NTR triggers a self-immolative fragmentation (1,6-elimination) and “turn on“NIR fluorescence. The reactive 1,4-aza quinone methide by-product is scavenged by water to generate a stable alcohol. (b) Chemical structures of heptamethine cyanine probes and key synthetic intermediates.

Scheme 2:

Synthesis of heptamethine cyanine dyes 5–10

Figure 1:

Spectra for dyes 4 and 10 (5 µM) in methanol. (a) Absorption spectra with vertical line showing absorbance at 808 nm, (b) Fluorescence spectra and emission counts integrated over acquisition range of 818 – 1000 nm, λ_ex = 808 nm, slit width = 5 nm.

Figure 2.

(a and c) Absorption spectra, (b and d) fluorescence spectra of 5 or 7 (10 μM) in PBS (pH 7.4) supplemented with NADH (1000 μM), before and 2 hour after addition of nitroreductase (NTR, 10 μg mL⁻¹). For 5 and 7, λ_ex = 750 nm.

Figure 3:

(a) Absorption spectra, (b) fluorescence spectra, (c) fluorescence intensity over time at 810 nm, for solutions of probe 9 (5 µM) before and after addition of NADH + NTR. (d) Fluorescence response of 9 to added analytes after 1 hour at 37 °C : (1) no added analyte, (2) Cysteine (1 mM), (3) Vitamin C (1 mM), (4) NaCl (1 mM), (5) Pig Liver esterase (10 μg/mL) (6) Glucose (1 mM), (7) Tyrosine (1 mM), (8) Butyrylcholinesterase (10 μg/mL), (9) NADH ( 500 μM), (10) Serine (1 mM), (11) Tryptophan (1 mM), (12) NADH (500 μM) + NTR (1 μg/mL). In all cases, λ_max = 810 nm, λ_max = 750 nm.

Figure 4:

Quantification of intracellular NIR fluorescence intensities as mean pixel intensity (MPI). A549 cells were incubated under conditions of normoxia (20% O₂) or hypoxia (1% O₂) for 24 hours, treated for 2 hours with either probe 5, 7, or 9; then epifluorescence images were acquired using an ICG filter (ex: 769/41, em: 832/37) Bars represent mean ±SD for triplicates, *p=0.0001, **p=0.0029, ***p<0.0001

Figure 5:

(a) NIR images of two Eppendorf tubes inside an animal in vivo imaging station (IVIS); ex: 745 nm, em: 850 nm. Left tube contained a solution of probe 9 (5 µM) + 500 µM NADH, the right tube is 1 hour after the addition of 1 µg/mL NTR to a solution of probe 9 (5 µM) + 500 µM NADH. The yellow oval identifies the region of interest that was used to calculate the Contrast to Background Ratio (CBR). (b) NIR fluorescence images of two mouse phantoms. The fillable port corresponding to the heart was filled with 5 μM of 9 + 500 μM NADH + 1 μg/mL NTR in 1X PBS Buffer pH 7.4. The imaging was conducted after 1 hour, a time period that allowed complete NTR catalyzed conversion of 9 into fluorescent product 10. (left) Mouse phantom open and exposed to the camera; (right) Mouse phantom covered with 0.5 cm thick chicken breast meat. The yellow circle in each image identifies the region of interest that was used to calculate the CBR.